Method for excision of plant embryos for transformation

ABSTRACT

This invention describes a simple method useful for the excision and isolation of maize immature embryos. The embryos are useful for plant tissue culture and transformation methods.

REFERENCE TO PRIOR APPLICATION

This application claims benefit of U.S. Provisional Application Ser. No.60/493,011, filed Aug. 5, 2003, which is incorporated herein byreference.

FIELD OF THE INVENTION

Disclosed herein is a method for the vacuum excision of explant tissuefrom plants. The method is particularly useful for isolating immatureembryogenic tissue for propagation and regeneration of a plant. Moreparticularly, the method is useful for the production of a transgenicplant.

The preparation of embryogenic tissue for plant propagation,regeneration and transformation is time consuming and labor intensive,especially as it involves manual excision of desired explant tissue. Forexample, in corn the manual removal of individual immature embryos is acommon means for isolating tissue useful for experiments. The manualexcision of embryogenic tissues is not only laborious, it is fraughtwith ergonomic issues. It would be of great benefit to the art of plantpropagation, regeneration and transformation to have a method ofexcising embryogenic tissue that is rapid and reduces ergonomic burdenon the user.

An object of this invention is to provide a simple method for theisolation of explant tissue. More particularly, the invention provides amethod directed to the use of a vacuum to isolate individual immatureembryos from an ear of corn.

SUMMARY OF THE INVENTION

This invention provides a simple method for the vacuum excision of aplant embryo. The method is useful for isolating immature maize embryosfrom ears of corn.

Vacuum excision may be carried out using an aspirator which, in itssimplest terms, is an isolation tube connected to a collectingreceptacle having a vacuum source. The aspirator is used to vacuumexcise and collect an immature embryo from a kernel, for example, theisolation of a maize immature embryo from a kernel. In one embodiment,the maize kernel is on an ear. Each immature embryo may be intact orpartial and may be accompanied by endosperm material. Each immatureembryo, either partial or intact, is useful for the production of callustissue and the regeneration of a fertile maize plant. Vacuum excision isalso used to isolate a slurry comprising both partial and intactimmature embryos. The slurry is useful for the production of callustissue and regeneration of a fertile maize plant.

Each immature embryo, either partial or intact, or the slurry comprisingboth partial and intact immature embryos, is also useful fortransformation. An intact immature embryo may be directly transformedshortly after excision, propagated and regenerated into a transgenicplant. Alternatively, an intact immature embryo, partial immatureembryo, or slurry comprising both partial and intact immature embryos,may be propagated into callus material that may be used fortransformation and regenerated into a fertile, transgenic maize plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an illustration of an aspirator useful for vacuum excision ofimmature embryos.

FIG. 2 is an illustration of an in-line sieving unit. FIG. 2A shows theunit in one piece and FIG. 2B shows the sections of the unit.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a novel method for the removal of an embryogenictissue explant for the propagation of tissue and regeneration of aplant. Preferably, the method is used with monocot plants including, butnot limited to, maize, wheat and rice. In particular, the method is usedfor the isolation of tissue for use with transformation methods. Themethod is particularly useful for the isolation of immature embryos(IEs) from any variety of maize, for the propagation of tissue andregeneration of a maize plant, and more particularly, for the productionof a stably transformed maize plant.

As used herein “tissue explant” or “explant tissue” is a first tissue,such as an immature embryo, isolated away from a second tissue, such asa corn kernel, wherein the isolated tissue is useful for the propagationof a plant cell, the maintenance of callus and/or for the regenerationof a plant. The explant or a cell derived therefrom may be used fortransformation purposes and may be transformed by any planttransformation method, preferably by Agrobacterium or particlebombardment. As used herein “propagation” is the maintenance of viableplant cells on a media, e.g. a callus tissue on a solid medium orprotoplasts in liquid medium. During propagation, the plant cells areusually dividing and producing an increasing number of cells or amountof tissue.

As used herein “regeneration” means the process of growing a plant froma plant cell (e.g., plant protoplast, callus or immature embryo). It iscontemplated that any cell from which a fertile plant may be regeneratedis useful for propagation or as a recipient cell for transformation.Callus may be initiated from tissue sources including, but not limitedto, immature embryos, seedling apical meristems, microspores and thelike. Those cells that are capable of proliferating as callus also arerecipient cells for genetic transformation. Preferably, the plant isfertile when fully regenerated.

For purposes of this invention, “kernel” means a tissue of a monocotplant that comprises an immature embryo along with endosperm tissue, forexample a maize kernel or a grain of wheat or rice. Typically, there isa single immature embryo per kernel. In maize, kernels are located onwhat is termed an ear; in wheat or rice, kernels are located on what istermed a head. Immature embryos are typically isolated from maize atapproximately 10 to 14 days post pollination and may range in size fromabout 1 mm to about 4 mm in the largest dimension (commonly called“length”). Isolated immature embryos of about 1.5 mm to about 2.5 mm arepreferred for use in many plant propagation and transformation methods.

An intact immature embryo is an embryo which, when isolated from akernel, is whole in nature; it comprises all of its natural parts in onepiece, organized as they naturally occur in the kernel. A partialimmature embryo is an embryo which, when isolated from a kernel, is notwhole in nature. For example, the partial immature embryo may be brokeninto one or more pieces during the excision process and may not compriseall of its natural parts in one piece. As used herein, a tissue is saidto be “embryogenic” if the cells of the tissue are capable of celldivision such that the amount of tissue increases and/or the tissue thatis formed is embryonic tissue. Immature embryos are an example of anembryogenic tissue.

The vacuum excision of the invention may be carried out using any typeof suitable vacuum excision apparatus such as the exemplary device shownin FIG. 1. Referring to FIG. 1, the size of the collecting receptacle 1used to collect the immature embryos may vary; in one embodiment thecollecting receptacle 1 is a 125 ml side-arm flask. A first piece oftubing 2 a is used for connecting an isolation tube 3 to the collectingreceptacle 1 and a second piece of tubing 2 b is used for connecting thecollecting receptacle 1 to the vacuum source 4. The pieces of tubing 2 aand 2 b may vary in diameter and length to accommodate excision of animmature embryo from a monocot kernel. In one embodiment, a useful innerdiameter of tubing 2 a and 2 b is about 6 mm. Attached to the firstpiece of tubing 2 a is the isolation tube 3 for extracting the immatureembryo. Tubing 2 a used to connect the collecting receptacle 1 to theisolation tube 3 can be any convenient length; for example, about 34 cmto about 42 cm is useful.

The isolation tube 3 is preferably a tube with a tapered end; morepreferably the isolation tube 3 used to extract the embryos is awide-bore pipette tip. The diameter of aperture of the isolation tube 3may vary; in one embodiment, the aperture diameter of the isolation tube3 may range from about 1.5 mm to about 4 mm, preferably an isolationtube 3 with an aperture diameter of about 2.5 mm is used. The apertureof the isolation tube 3 should be varied to accommodate the size of theembryos being isolated, with a larger aperture being used to isolatelarger embryos. For example, an isolation tube 3 with an aperturediameter of about 2.5 mm can be used to isolate immature embryos ofabout 2.5 mm or smaller.

The amount of vacuum applied from a vacuum source 4 to the collectingreceptacle 1 may vary; any vacuum that causes the excision and removalof the immature embryo from the kernel is within the scope of theinvention. Useful vacuum may range from about 500 to about 740 mm Hg;preferably from about 525 to about 740 mm Hg, even more preferably fromabout 550 to about 740 mm Hg; most preferably, about 610 to about 740 mmHg is used to isolate embryos. One skilled in the art would know that itwould be possible to change the size of the collecting receptacle 1, thediameter of the tubing 2 a and 2 b, the length of the tubing 2 a and 2b, the diameter or length of the isolation tube 3 or pipette tip used toextract the embryos, and the vacuum applied to achieve vacuum isolationof immature embryos from maize.

In one embodiment, the immature embryos are drawn into a collectingreceptacle 1 into a liquid solution such as cell growth media.Alternatively, the embryos are drawn into a collecting receptacle 1fitted to contain a support to catch the embryos. This support may be asolid or semi-solid medium, felt, cotton packing, mesh, a sieve or acombination of supports to catch the embryos as they are received intothe collecting receptacle 1. In another embodiment, the embryos aredrawn up into the isolation tube 3 and, rather than be collected in thecollecting receptacle 1, the vacuum is reversed and pressure is used topush the embryos out for collection, for example, onto a plate of solidor semi-solid medium.

With reference to FIGS. 2A and 2B, an in-line sieving unit 5 is placedin the line of the first piece of tubing 2 a connecting the isolationtube 3 to the collecting receptacle 1 such that embryos are collected ona mesh or sieving material rather than in the collecting receptacle 1and retrieved therefrom. In one embodiment, several layers of mesh areused in the in-line sieving unit 5, with the mesh size decreasing withdistance from the isolation tube 3. In one embodiment, the in-linesieving unit 5 is assembled from sections of nominal two inch tubing andcomprises three layers of autoclavable, stainless steel sievingmaterial, each layer being separated by approximately 1.3 cm and thelayers of sieving material are joined to the tubing 2 a via connectors 9a and 9 b. In one embodiment, the layer of sieving material 6 nearest tothe isolation tube 3 has openings of about 1130 microns, the middlelayer of sieving material 7 has openings of about 979 microns and thelayer of sieving material 8 has openings of about 472 microns. Embryosare collected on any layer of the sieving materials 6 and 7 and 8 andare manually retrieved or washed off the sieving material 6 and 7 and 8by a gentle stream of liquid, such as media, for further use. In anotherembodiment, the layers of sieving material 6 and 7 and 8 may be alteredsuch that only one or two layers are used, or that four or more layersare used, or that any two layers are of the same type of sievingmaterial. One skilled in the art would know that the diameter or lengthof the in-line sieving unit 5, position within the tubing 2 a, openingsizes of the sieving material 6 and 7 and 8 and number of sievingmaterial layers 6 and 7 and 8 may be varied and still achieve vacuumisolation of immature embryos from maize.

It is believed that this method may be applicable to all kinds of plantsfor the isolation of one tissue from another, including but not limitedto separating a first dicot tissue from a second dicot tissue or a firstmonocot tissue from a second monocot tissue. For example, vacuumisolation is useful to simply isolate the seeds of a plant from a floralstructure, such as, but not limited to, the isolation of mature seedsfrom a mixture of soybean seeds and seed pods. In another example,vacuum isolation is useful to isolate embryonic tissue from othertissues. Alternatively, vacuum excision is useful to separate mixedtissues contained within a slurry, such as the isolation of monocotimmature embryos from a slurry comprising immature embryos, endospermand various other kernel tissues. As exemplified in the presentinvention, vacuum isolation is useful to isolate maize immature embryosfrom kernels on an ear.

Recombinant DNA Constructs—When transformation of the tissue explant iscarried out, the present invention contemplates the use of any DNAmolecule capable of imparting any desired attribute to a plant,including but not limited to herbicide resistance or tolerance, insectresistance or tolerance, disease resistance or tolerance (viral,bacterial, fungal, nematode), stress tolerance and/or resistance, asexemplified by resistance or tolerance to drought, heat, chilling,freezing, excessive moisture, salt stress and oxidative stress,increased yield, food or feed content and value, physical appearance,male sterility, drydown, standability, prolificacy, starch quantity andquality, oil quantity and quality, protein quality and quantity, aminoacid composition, and the like.

Genes for imparting such desired characteristics are assembled inrecombinant DNA constructs using methods known to those of ordinaryskill in the art. Standard cloning techniques are useful for buildingDNA constructs and vectors suitable for use in the transformation ofplant cells. One such technology for building DNA constructs and vectorsfor transformation is the GATEWAY™ cloning technology (available fromInvitrogen Life Technologies, Carlsbad, Calif.) which is disclosed inU.S. Pat. Nos. 5,888,732 and 6,277,608, and U.S. Patent ApplicationPublications 2001283529, 2001282319 and 20020007051, all of which areincorporated herein by reference. The GATEWAY™ Cloning TechnologyInstruction Manual, which is supplied by Invitrogen, also providesconcise directions for routine cloning of any desired DNA into a vectorcomprising operable plant expression elements.

As used herein, “exogenous DNA” refers to DNA which does not naturallyoriginate from the particular construct, cell or organism in which thatDNA is found. Recombinant DNA constructs used for transforming plantcells will comprise exogenous DNA and usually other elements asdiscussed below. As used herein “transgene” means an exogenous DNA thathas been incorporated into a host genome or is capable of autonomousreplication in a host cell and is capable of causing the expression ofone or more cellular products. Exemplary transgenes will provide thehost cell, or plants regenerated therefrom, with a novel phenotyperelative to the corresponding non-transformed cell or plant. Transgenesmay be directly introduced into a plant by genetic transformation, ormay be inherited from a plant of any previous generation that wastransformed with the exogenous DNA.

As used herein “gene” or “coding sequence” means a DNA sequence fromwhich an RNA molecule is transcribed. The RNA may be an mRNA thatencodes a protein product, an RNA which functions as an anti-sensemolecule, or a structural RNA molecule such as a tRNA, rRNA, or snRNA,or other RNA. As used herein “expression” refers to the combination ofintracellular processes, including transcription and translation,undergone by a DNA molecule, such as a structural gene to produce apolypeptide, or a non-structural gene to produce an RNA molecule.

As used herein “promoter” or “5′ regulatory region” means a region ofDNA sequence that is essential for the initiation of transcription ofRNA from DNA. Promoters are located upstream of DNA to be translated,have regions that act as binding sites for RNA polymerase and haveregions that work with other factors to promote RNA transcription.

As is well known in the art, recombinant DNA constructs typically alsocomprise other regulatory elements in addition to a promoter, such asbut not limited to 3′ untranslated regions (such as polyadenylationsites), transit or signal peptides and marker genes elements. Forinstance, see U.S. Pat. No. 6,437,217 which discloses a maize RS81promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter,U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S.Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No.6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357which discloses a rice actin 2 promoter and intron, U.S. Pat. No.5,837,848 which discloses a root specific promoter, U.S. Pat. No.6,084,089 which discloses cold inducible promoters, U.S. Pat. No.6,294,714 which discloses light inducible promoters, U.S. Pat. No.6,140,078 which discloses salt inducible promoters, U.S. Pat. No.6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No.6,175,060 which discloses phosphorus deficiency inducible promoters,U.S. Patent Application Publication 2002/0192813A1 which discloses 5′,3′ and intron elements useful in the design of effective plantexpression vectors, U.S. patent application Ser. No. 09/078,972 whichdiscloses a coixin promoter, and U.S. patent application Ser. No.09/757,089 which discloses a maize chloroplast aldolase promoter, all ofwhich are incorporated herein by reference.

In some aspects of the invention it is preferred that the promoterelement in the DNA construct should express in a constitutive manner. Inother aspects, it is preferred that the promoter element in the DNA becapable of causing sufficient expression to result in the production ofan effective amount of a desired gene under particular conditions, forexample, at a particular time in development or in a particular tissue.By avoiding continuous high-level expression of transgenes, anyundesired effects caused by continual over-expression of transgenes, orectopic expression in various tissues or at various times, can beminimized or eliminated.

During transformation, exogenous DNA may be introduced randomly, i.e. ata non-specific location, in the plant genome. In some cases, it may beuseful to target heterologous DNA insertion in order to achievesite-specific integration, e.g. to replace an existing gene in thegenome. In some other cases it may be useful to target a heterologousDNA integration into the genome at a predetermined site from which it isknown that gene expression occurs. Several site-specific recombinationsystems exist which are known to function in plants include Cre/lox asdisclosed in U.S. Pat. No. 4,959,317 and FLP/FRT as disclosed in U.S.Pat. No. 5,527,695, both incorporated herein by reference.

Constructs and vectors may also include a transit peptide for targetingof a gene target to a plant organelle, particularly to a chloroplast,leucoplast or other plastid organelle. For a description of the use of achloroplast transit peptide see U.S. Pat. No. 5,188,642, incorporatedherein by reference.

In practice DNA is introduced into only a small percentage of targetcells in any one experiment. Marker genes are used to provide anefficient system for identification of those cells that are stablytransformed by receiving and integrating a transgenic DNA construct intotheir genomes. Preferred marker genes provide selective markers thatconfer resistance to a selective agent, such as an antibiotic orherbicide. Potentially transformed cells are exposed to the selectiveagent. In the population of surviving cells will be those cells where,generally, the resistance-conferring gene has been integrated andexpressed at sufficient levels to permit cell survival. Cells may betested further to confirm stable integration of the exogenous DNA.Useful selective marker genes include those conferring resistance toantibiotics such as kanamycin (nptII), hygromycin B (aph IV) andgentamycin (aac3 and aacC4) or resistance to herbicides such asglufosinate (bar or pat) and glyphosate (EPSPS; CP4). Examples of suchselectable markers are illustrated in U.S. Pat. Nos. 5,550,318;5,633,435; 5,780,708 and 6,118,047, all of which are incorporated hereinby reference. Screenable markers which provide an ability to visuallyidentify transformants can also be employed, e.g., a gene expressing acolored or fluorescent protein such as a luciferase or green fluorescentprotein (GFP) or a gene expressing a beta-glucuronidase or uidA gene(GUS) for which various chromogenic substrates are known.

Protein and Polypeptide Molecules—Polypeptides of the present inventionthat represent whole proteins or at least a sufficient portion of theentire protein to impart the relevant biological activity of theprotein, e.g. increased yield or increased tolerance to water-deficit.The term “protein” also includes molecules consisting of one or morepolypeptide chains. Thus, a polypeptide useful in the present inventionmay constitute an entire gene product or one or more functional portionsof a natural protein that provides a desired agronomic trait.

Homologs of the polypeptides imparting a desired trait or phenotype maybe identified by comparison of the amino acid sequence of thepolypeptide to amino acid sequences of polypeptides from the same ordifferent plant sources, e.g. manually or by using known homology-basedsearch algorithms such as those commonly known and referred to as BLAST,FASTA, and Smith-Waterman.

A further aspect of the invention comprises functional homologousproteins which differ in one or more amino acids from those of a firstpolypeptide provided herein as the result of one or more of thewell-known conservative amino acid substitutions, e.g. valine is aconservative substitute for alanine and threonine is a conservativesubstitute for serine. When such a homologous protein is expressed in atransgenic plant, the homologous protein will affect the transgenicplant in a substantially equivalent manner as the first polypeptide.

Transformation Methods and Transgenic Plants—Methods and compositionsfor transforming plants by introducing an exogenous DNA into a plantgenome in the practice of this invention can include any of thewell-known and demonstrated methods. Preferred methods of planttransformation are microprojectile bombardment as illustrated in U.S.Pat. Nos. 5,015,580; 5,550,318; 5,538,880; 6,160,208; 6,399,861 and6,403,865 and Agrobacterium-mediated transformation as illustrated inU.S. Pat. Nos. 5,635,055; 5,824,877; 5,591,616; 5,981,840 and 6,384,301,all of which are incorporated herein by reference. Immature embryos thatare useful in the art of transformation are isolated approximately 10 to14 days post-pollination. The method is also useful for the isolation ofembryos from ears aged 15 days or greater post-pollination.

Transformation methods of this invention to provide plants with anydesired phenotype resulting from transgene expression are preferablypracticed in tissue culture on media and in a controlled environment.“Media” refers to the numerous liquid, solid, or semi-solid nutrientmixtures that are used to grow cells in vitro, that is, outside of theintact living organism. Recipient cell targets include, but are notlimited to, meristem cells, callus, immature embryos and gametic cellssuch as microspores, pollen, sperm and egg cells. One skilled in the artwould know that the type of media and times of transfer used forpropagation and regeneration may vary yet still produce identical orsubstantially identical results.

As used herein a “transgenic” organism is one whose genome has beenaltered by the incorporation of foreign genetic material or additionalcopies of native genetic material, e.g. by transformation orrecombination. As used herein “transgenic plant” means a plant orprogeny plant of any subsequent generation derived therefrom, whereinthe DNA of the plant or progeny thereof contains an introduced exogenousDNA not originally present in a non-transgenic plant of the same strain.The transgenic plant may additionally contain sequences that are nativeto the plant being transformed, but wherein the exogenous DNA has beenaltered in order to change the level or pattern of expression of thegene.

As used herein an “R_(o) transgenic plant” is a plant which has beendirectly transformed with an exogenous DNA or has been regenerated froma cell or cell cluster which has been transformed with an exogenous DNA.As used herein “progeny” means any subsequent generation, including theseeds and plants therefrom, which is derived from a particular parentalplant or set of parental plants; the resultant progeny line may beinbred or hybrid. Progeny of a transgenic plant of this invention canbe, for example, self-crossed, crossed to a transgenic plant, crossed toa non-transgenic plant, and/or back crossed. As used herein “cropplants” of interest include, but are not limited to soy, cotton, canola,maize, wheat, sunflower, sorghum, alfalfa, barley, millet, rice,tobacco, fruit and vegetable crops, and turf grass.

The seeds of this invention can be harvested from fertile transgenicplants and be used to grow progeny generations of plants of thisinvention including a hybrid plant line.

EXAMPLES

Having now generally described the invention, the same will be morereadily understood through reference to the following examples that areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

Example 1

This example illustrates the use of vacuum excision to isolate maizeimmature embryos and the production of callus tissue from the isolatedembryos.

Two Zea mays lines, a male line and a female line, were prepared forvacuum excision. Ears were collected 10–12 days post pollination andwere sterilized by immersion in 80% ethanol. Following a shortair-drying, each ear was placed on sterilized aluminum foil and gentlyrolled on a flat surface. A scalpel was used to slice off the crowns(tops) of the kernels.

Using a sterile vacuum aspirator (see FIG. 1), the immature embryos(IEs) and some surrounding endosperm tissue was excised using a vacuumof about 610 mm Hg by introducing the pipette tip into the apical end ofthe kernel with a horizontal position of the wide bore pipette tip.Occasionally, sterile medium (e.g., 211 liquid media) was also drawninto the aspirator to propel any bits of tissue in the tubing into thecollecting receptacle.

Isolation of Partial and Intact Immature Embryos by Vacuum Isolation

When the excision was completed, the liquid and tissue in the collectionvessel was poured out onto solid media and excess solution removed byaspiration. The intact immature embryos were isolated and arrangedscutellar side up on solid media. Two types of control experiments werecarried out; intact immature embryos were isolated manually using small,sterile spatulas and either A) placed directly onto solid media or B)placed into liquid media and then placed onto solid media. The isolatedintact IEs of the male line were placed onto solid 211 media. Theisolated intact IEs of the female line were placed onto solid 851 media.The intact IEs were incubated at approximately 27–28° C. and callusformation was observed at about 8–11 days after excision. Callus wastransferred to fresh media as needed to maintain growth. Exemplary mediaare listed in Table 1.

Callus was initiated from vacuum excised explants from both maize lines.Data are shown in Table 2 and Table 3, comparing the callus responsefrom the manually excised versus vacuum excised immature embryos for thetwo maize lines. As can be seen from the data, a number of vacuumexcised explants from both maize lines resulted in the formation ofcallus tissue.

In other experiments, when the excision was completed, the liquid andtissue in the collection vessel was poured out into a thin layer ontosolid media and excess solution (about 2 ml) was removed. Partialimmature embryos were selected for callus production. The isolatedpartial IEs from the male maize line were placed onto solid 211 mediaand incubated at approximately 28° C. The isolated partial IEs from thefemale maize line were placed onto solid 851 media and incubated atapproximately 28° C. Callus formation for each line was observed atabout 8–11 days after excision. Callus was transferred to fresh media asneeded to maintain growth. Exemplary media are listed in Table 1.

TABLE 1 Media useful for tissue propagation and regeneration Media TypeComponents pH Gelling agent* 190   1X MS salts   1X MS Fromm vitamins;0.15 g/L L- 5.8   6 g/L Phytagar Asparagine; 0.1 g/L myo-inositol; 10g/L glucose; 20 g/L maltose; 250 mg/L carbenicillin; 100 uM glyphosate192   1X MS salts   1X MS Fromm vitamins; 3.52 mg/L 5.6   6 g/L PhytagarBAP; 50 mg/L casamino acids; 1.36 g/L proline; 30 g/L sucrose; 250 mg/Lcarbenicillin; 100 uM glyphosate 211   1X N6 basal   1 mg/L2,4-dichlorophenoxyacetic acid 5.8   2 g/L Gelgro agar salts (2–4,D); 1mg/L thiamine; 0.5 mg/L nicotinic acid; 0.91 g/L L-asparaginemonohydrous; 100 mg/L myo-inositol; 0.5 g/L 2-(4-morpholino)-ethanesulfonic acid (MES); 1.6 g/L MgCL2.6H2O; 100 mg/L casein hydrolysate;0.69 g/L proline; 20 g/L sucrose; 16.9 mg/L silver nitrate 217   1X N6basal 1.0 mg/L Thiamine, 0.5 mg/L Nicotinic 5.8   6 g/L Phytagar saltsAcid; 3.52 mg/L BAP; 0.91 g/L asparagine; 0.1 g/L myo-inositol; 0.5 g/L2-(4-morpholino)-ethane sulfonic acid (MES); 1.6 g/L MgCL2.6H2O; 100mg/L casein hydrolysate; 0.69 g/L proline; 20 g/L sucrose 600 0.5X MSsalts   1X MS vitamins (without myo-inositol); 5.2 5.5 g/L low melt 0.5mg/L thiamine HCl; 0.115 g/L agarose proline; 10 g/L glucose; 20 g/Lsucrose; 3 mg/L 2,4-D; 20 μM silver nitrate; 200 μM acetosyringone 6010.5X MS salts 0.5X MS vitamins (without myo- 5.4 none inositol); 0.115g/L proline; 26 g/L glucose; 68.5 g/L sucrose 632   1X MS salts   1X MSFromm vitamins; 0.05 g/L myo- 5.8   6 g/L Phytagar inositol; 60 g/Lsucrose; 250 mg/L carbenicillin; 100 mg/L paromomycin 850   1X MS salts  1X MS vitamins; 0.5 mg/L thiamine 5.8   3 g/L phytagel HCl; 1.38 g/Lproline; 30 g/L sucrose; 0.5 g/L casein hydrolysate; 0.5 mg/L 2,4-D;0.01 mg/L BAP; 100 uM glyphosate; 500 mg/L carbenecillin; 20 uM silvernitrate 851   1X MS salts   1X MS vitamins; 1.38 g/L proline; 30 g/L 5.83.0 g/L phytagel sucrose; 0.5 g/L casein hydrolysate; 0.5 mg/L 2,4-D;2.2 mg/L picloram; 20 uM silver nitrate 852 0.5X MS salts 0.5X MSvitamins; 20 g/L sucrose; 0.5 mg/L 5.8   3 g/L phytagel IBA; 0.5 mg/L1-naphthalene acetic acid (NAA); 100 uM glyphosate *Liquid media havethe same components as solid media with the exception of a gellingagent. Filtered, reverse-osmosis water was used to prepare media used inthe practice of this invention.

TABLE 2 Callus production from vacuum excised explants from a maize maleline I.E. Size # of # IE's Method (mm) Kernels Excised # Calli ManualExcision 2.0–2.2 100 98 95 Manual Excision 1.8–2.0 95 88 83 ManualExcision 1.7–1.9 107 90 77 Manual Excision into Liquid 2.0–2.2 92 84 48Manual Excision into Liquid 1.8–2.0 100 90 53 Manual Excision intoLiquid 1.7–1.9 90 72 31 Vacuum Excision 2.0–2.2 98 39 16 Vacuum Excision1.8–2.0 100 63 40 Vacuum Excision 1.7–1.9 100 54 7

TABLE 3 Callus production from vacuum excised explants from a maizefemale line I.E. Size # of # IE's Method (mm) Kernels Excised # CalliManual Excision 1.4–1.6 97 88 88 Manual Excision 1.3–1.8 95 85 79 ManualExcision 1.7 74 45 42 Manual Excision into Liquid 1.4–1.6 73 62 62Manual Excision into Liquid 1.3–1.8 72 56 54 Manual Excision into Liquid1.7 65 35 33 Vacuum Excision 1.4–1.6 84 34 28 Vacuum Excision 1.3–1.8 8358 51 Vacuum Excision 1.7 80 6 5

Isolation of Intact and Partial Immature Embryos using a Sieving Unit

Immature embryos of the female maize line were isolated using vacuumexcision wherein the aspirator was fitted with an in-line sieving unit.Following excision of the embryos from the ear, approximately 10–15 mlsof liquid (e.g., liquid media) were also drawn through the in-linesieving unit to ensure any material in the isolation tube was drawn ontothe sieving materials. The in-line sieving unit was then disassembledand the immature embryos and other material on the sieving material wereflushed off and collected in a petri dish. The embryos, both partial andintact, were then transferred to solid 851 media and callus wasproduced.

Isolation of an Immature Embryo Slurry by Vacuum Isolation

A “slurry” of immature embryos of a female maize line, comprising bothpartial and intact IEs as well as any other material collected from thekernels, was also isolated using vacuum excision, both with and withoutthe in-line sieving unit. When the excision was completed without thesieving unit, approximately 1–2 ml of the liquid and tissue in thecollection receptacle, that is, the slurry comprising intact and partialimmature embryos, was plated out into a thin layer (thin-plated) ontosolid 851 media and incubated at approximately 28° C. When excision wascompleted using the sieving unit, the unit was disassembled and thecollected tissue was released by swirling the sieving layers in liquidmedia, releasing the trapped tissues and creating a slurry comprisingboth partial and intact IEs as well as any other material collected fromthe kernels. The slurry was then thin-plated onto solid 851 media asdescribed.

Using both types of isolation methods, callus formation for each linewas observed at about 8–11 days after excision.

Callus prepared from intact IEs, partial IEs or a slurry mixture ofimmature and mature IEs is regenerated into fertile plants using mediaand methods of transfer known to those in the art (see also Example 2).

Example 2

This example describes the production of a transgenic plant prepared bytransformation of immature embryos isolated using vacuum excision.

Transformation of Intact Immature Embryos

Intact immature embryos were vacuum excised (˜610 mm Hg) from a femalemaize line ears 10–12 days post pollination as described in Example 1and used for Agrobacterium mediated transformation.

Agrobacterium was transformed to contain a vector comprising a greenfluorescent protein (GFP) marker gene and a CP4 marker gene allowing forherbicide resistance. Excess liquid was removed from the vacuum isolatedintact embryos. The embryos were allowed to incubate with theAgrobacterium for about 15–20 minutes at room temperature. The intactembryos were removed from the Agrobacterium solution, placed onto solid600 media and allowed to incubate overnight. Following this, the vacuumexcised tissues were placed onto solid 850 media for about 3 weeks,followed by about 1 week on solid 192 media, followed by about 2 weekson solid 190 media, followed by about 3 weeks on solid 190 media, andthen a transfer to soil.

Eleven different transgenic events from the vacuum excised tissues wereselected for regeneration and one stable transgenic plant exhibiting GFPexpression was obtained from the vacuum excised transformed tissue. Theplant was allowed to mature and seed was recovered.

Transformation of a Mixture Immature Embryos

Partial and intact immature embryos were vacuum excised (˜610 mm Hg)from a female maize line ears 10–12 days post pollination as describedin Example 1 and used for Agrobacterium mediated transformation. Themixture of partial and intact IEs are exposed to Agrobacterium asdescribed above and both intact and partial embryos are removed from theAgrobacterium solution and placed into the selection and regenerationprocesses. Transgenic plantlets are recovered, allowed to mature intofertile transgenic plants and seed may be collected.

Example 3

This example describes the production of a transgenic plant prepared bytransformation of callus material prepared from immature embryosisolated using vacuum excision. Two Zea mays lines, a representativemale and female line, were prepared for vacuum excision as described inExample 1.

Intact Immature Embryos

Callus isolated from intact immature embryos is useful fortransformation and regeneration of mature transgenic plants. Intactimmature embryos were vacuum excised into the collecting receptacle, andthe liquid and tissue in the collection vessel was poured out into athin layer onto solid media. In other experiments, the sieving unit wasused to collect the intact IEs. Following either excision method, intactimmature embryos were selected for callus production. The isolated IEsfrom the male maize line were placed onto solid 211 media and incubatedat approximately 28° C. The isolated IEs from the female maize line wereplaced onto solid 851 media and incubated at approximately 28° C. Callusformation for each line was observed at about 8–11 days after excision.Callus derived from intact IEs is useful for transformation,regeneration and production of fertile, transgenic plants.

Partial Immature Embryos

Callus isolated from partial immature embryos is also useful fortransformation and regeneration of mature transgenic plants. Partialimmature embryos were vacuum excised either directly into the collectingreceptacle or, in some experiments, the IEs were collected on thesieving unit. The partial IEs isolated from either method were used forcallus production. Callus prepared from partial IEs was subcultured andgrown to increase the amount of tissue useful for transformation and/orregeneration into mature transgenic plants. The callus tissue wasexposed to Agrobacterium comprising genes of interest for transfer tothe recipient genome. Following culturing and selection on mediaappropriate for the maize line employed, transformed cells are selectedand plantlets will be regenerated. The plantlets are allowed to matureand transgenic seed may be collected.

Intact and Partial Immature Embryos

In other experiments, the material used for transformation was derivedfrom a slurry of intact and partial immature embryos. Agrobacterium wastransformed to contain various vectors comprising an NPTII marker geneallowing for paromomycin resistance. Callus derived from the slurry ofintact and partial immature embryos was exposed to the Agrobacterium fortransformation. Callus exhibiting resistance to paromomycin wasrecovered and regenerated into plantlets, with greater than 10% of thecallus forming transgenic plantlets. The transgenic plantlets are grownunder greenhouses into mature plants and seed may be collected.

1. A method for isolating an immature maize plant embryo from a maizekernel comprising: a) exposing a maize endosperm containing an immaturemaize embryo by cutting the kernel; b) excising said immature maizeembryo by aspiration; and c) collecting said excised immature maizeembryo.
 2. The method as in claim 1 wherein said immature embryo isintact.
 3. The method of claim 1 wherein said immature embryo ispartial.
 4. The method of claim 1 wherein a plurality of immatureembryos are isolated from a number of kernels on an ear of maize.
 5. Themethod of claim 4 wherein the plurality of immature embryos is a mixtureof intact and partial embryos.
 6. The method as in claim 1 furthercomprising propagating the excised embryo obtained in step (c).
 7. Themethod of claim 6 wherein the propagating comprises the production ofcallus tissue.
 8. The method of claim 6 wherein the propagatingcomprises the regeneration of a plant to produce a regenerated plant. 9.The method of claim 8 wherein the regenerated plant is a fertile plant.10. The method of claim 9, further comprising obtaining seed from thefertile plant.
 11. The method of claim 10, further comprisingregenerating plants from the seed thus obtained.
 12. The method of claim1, further comprising propagating the excised embryo obtained in step(c) to form callus tissue.